The present invention relates to grapevine leafroll virus proteins, DNA molecules encoding these proteins, and their uses.
The world""s most widely grown fruit crop, the grape (Vitis sp.), is cultivated on all continents except Antarctica. However, major grape production centers are in European countries (including Italy, Spain, and France), which constitute about 70% of the world grape production (Mullins et al., Biology of the Grapevine, Cambridge, U.K.:University Press (1992)). The United States, with 300,000 hectares of grapevines, is the eighth largest grape grower in the world. Although grapes have many uses, a major portion of grape production (xcx9c80%) is used for wine production. Unlike cereal crops, most of the world""s vineyards are planted with traditional grapevine cultivars, which have been perpetuated for centuries by vegetative propagation. Several important grapevine virus and virus-like diseases, such as grapevine leafroll, corky bark, and Rupestris stem pitting, are transmitted and spread through the use of infected vegetatively propagated materials. Thus, propagation of certified, virus-free materials is one of the most important disease control measures. Traditional breeding for disease resistance is difficult due to the highly heterozygous nature and outcrossing behavior of grapevines, and due to polygenic patterns of inheritance. Moreover, introduction of a new cultivar may be prohibited by custom or law. Recent biotechnology developments have made possible the introduction of special traits, such as disease resistance, into an established cultivar without altering its horticultural characteristics.
Many plant pathogens, such as fungi, bacteria, phytoplasmas, viruses, and nematodes can infect grapes, and the resultant diseases can cause substantial losses in production (Pearson et al., Compendium of Grape Diseases, American Phytopathological Society Press (1988)). Among these, viral diseases constitute a major hindrance to profitable growing of grapevines. About 34 viruses have been isolated and characterized from grapevines. The major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus, other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) the rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, UN, Rome, Italy (1993)).
Of the major virus diseases, the grapevine leafroll complex is the most widely distributed throughout the world. According to Goheen (Goheen, xe2x80x9cGrape Leafroll,xe2x80x9d in Frazier et al., eds., Virus Diseases of Small Fruits and Grapevines (A Handbook), University of California, Division of Agricultural Sciences, Berkeley, Calif., USA, pp. 209-212 (1970) (xe2x80x9cGoheen (1970)xe2x80x9d), grapevine leafroll-like disease was described as early as the 1850s in German and French literature. However, the virus nature of the disease was first demonstrated by Scheu (Scheu, xe2x80x9cDie Rollkrankheit des Rebstockes (Leafroll of grapevine),xe2x80x9d D. D. Weinbau 14:222-358 (1935) (xe2x80x9cScheu (1935)xe2x80x9d)). In 1946, Harmon and Snyder (Harmon et al., xe2x80x9cInvestigations on the Occurrence, Transmission, Spread and Effect of xe2x80x98Whitexe2x80x99 Fruit Colour in the Emperor Grape,xe2x80x9d Proc. Am. Soc. Hort. Sci. 74:190-194 (1946)) determined the virus nature of White Emperor disease in California. It was later proven by Goheen et al. (Goheen et al., xe2x80x9cLeafroll (White Emperor Disease) of Grapes in California, Phytopathology, 48:51-54 (1958) (xe2x80x9cGoheen (1958)xe2x80x9d)) that both leafroll and xe2x80x9cWhite Emperorxe2x80x9d diseases were the same, and only the name xe2x80x9cleafrollxe2x80x9d was retained.
Leafroll is a serious virus disease of grapes and occurs wherever grapes are grown. This wide distribution of the disease has come about through the propagation of diseased vines. It affects almost all cultivated and rootstock varieties of Vitis. Although the disease is not lethal, it causes yield losses and reduction of sugar content. Scheu estimated in 1936 that 80 percent of all grapevines planted in Germany were infected (Scheu, Mein Winzerbuch, Berlin:Reichsnahrstand-Verlags (1936)). In many California wine grape vineyards, the incidence of leafroll (based on a survey of field symptoms conducted in 1959) agrees with Scheu""s initial observation in German vineyards (Goheen et al., xe2x80x9cStudies of Grape Leafroll in California,xe2x80x9d Amer. J. Enol. Vitic., 10:78-84 (1959)). The current situation on leafroll disease does not seem to be any better (Goheen, xe2x80x9cDiseases Caused by Viruses and Viruslike Agents,xe2x80x9d The American Phytopathological Society, St. Paul, Minn.:APS Press, 1:47-54 (1988) (xe2x80x9cGoheen (1988)xe2x80x9d). Goheen also estimated that the disease causes an annual loss of about 5-20 percent of the total grape production (Goheen (1970) and Goheen (1988)). The amount of sugar in individual berries of infected vines is only about xc2xd to ⅔ that of berries from noninfected vines (Goheen (1958)).
Symptoms of leafroll disease vary considerably depending upon the cultivar, environment, and time of the year. On red or dark-colored fruit varieties, the typical downward rolling and interveinal reddening of basal, mature leaves is the most prevalent in autumn; but not in spring or early summer. On light-colored fruit varieties however, symptoms are less conspicuous, usually with downward rolling accompanied by interveinal chlorosis. Moreover, many infected rootstock cultivars do not develop symptoms. In these cases, the disease is usually diagnosed with a woody indicator indexing assay using Vitis vivifera cv. Carbernet Franc (Goheen (1988)).
Ever since Scheu demonstrated that leafroll was graft transmissible, a virus etiology has been suspected (Scheu (1935)). Several virus particle types have been isolated from leafroll diseased vines. These include potyvirus-like (Tanne et al., xe2x80x9cPurification and Characterization of a Virus Associated with the Grapevine Leafroll Disease,xe2x80x9d Phytopathology, 67:442-447 (1977)), isometric virus-like (Castellano et al., xe2x80x9cVirus-like Particles and Ultrastructural Modifications in the Phloem of Leafroll-affected Grapevines,xe2x80x9d Vitis, 22:23-39 (1983) (xe2x80x9cCastellano (1983)xe2x80x9d) and Namba et.al., xe2x80x9cA Small Spherical Virus Associated with the Ajinashika Disease of Koshu Grapevine, Ann. Phytopathol. Soc. Japan, 45:70-73 (1979)), and closterovirus-like (Namba, xe2x80x9cGrapevine Leafroll Virus, a Possible Member of Closteroviruses, Ann. Phytopathol. Soc. Japan, 45:497-502 (1979)) particles. In recent years, however, long flexuous closteroviruses ranging from 1,400 to 2,200 nm have been most consistently associated with leafroll disease (FIG. 1) (Castellano (1983), Faoro et al., xe2x80x9cAssociation of a Possible Closterovirus with Grapevine Leafroll in Northern Italy,xe2x80x9d Riv. Patol. Veg., Ser IV, 17:183-189 (1981), Gugerli et al., xe2x80x9cL""enroulement de la vigne: mise en xc3xa9vidence de particules virales et developpement d""une mxc3xa9thode immuno-enzymatique pour le diagnostic rapide (Grapevine Leafroll: Presence of Virus Particles and Development of an Immuno-enzyme method for Diagnosis and Detection),xe2x80x9d Rev. Suisse Viticult. Arboricult. Hort., 16:299-304 (1984) (xe2x80x9cGugerli (1984)xe2x80x9d), Hu et al., xe2x80x9cCharacterization of Closterovirus-like Particles Associated with Grapevine Leafroll Disease,xe2x80x9d J. Phytopathol., 128:1-14 (1990) (xe2x80x9cHu (1990)xe2x80x9d), Milne et al., xe2x80x9cClosterovirus-like Particles of Two Types Associated with Diseased Grapevines,xe2x80x9d Phytopathol. Z., 110:360-368 (1984), Zee et al., xe2x80x9cCytopathology of Leafroll-diseased Grapevines and the Purification and Serology of Associated Closteroviruslike Particles,xe2x80x9d Phytopathology, 77:1427-1434 (1987) (xe2x80x9cZee (1987)xe2x80x9d), and Zimmermann et al., xe2x80x9cCharacterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine,xe2x80x9d J. Phytopathol., 130:205-218 (1990) (xe2x80x9cZimmermann (1990)xe2x80x9d)). These closteroviruses are referred to as grapevine leafroll associated viruses (xe2x80x9cGLRaVxe2x80x9d). At least six serologically distinct types of GLRaV""s (GLRaV-1 to -6) have been detected from leafroll diseased vines (Table 1) (Boscia et al., xe2x80x9cNomenclature of Grapevine Leafroll-associated Putative Closteroviruses, Vitis, 34:171-175 (1995) (xe2x80x9cBoscia (1995)xe2x80x9d) and (Martelli, xe2x80x9cLeafroll,xe2x80x9d pp. 37-44 in Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, Rome Italy, (1993) (xe2x80x9cMartelli Ixe2x80x9d)). The first five of these were confirmed in the 10th Meeting of the International Council for the Study of Virus and Virus Diseases of the Grapevine (xe2x80x9cICVGxe2x80x9d) (Volos, Greece, 1990).
Through the use of monoclonal antibodies, however, the original GLRaV II described in Gugerli (1984) has been shown to be an apparent mixture of at least two components, IIa and IIb (Gugerli et al., xe2x80x9cGrapevine Leafroll Associated Virus II Analyzed by Monoclonal Antibodies,xe2x80x9d 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Montreux, Switzerland, pp. 23-24 (1993) (xe2x80x9cGugerli (1993)xe2x80x9d)). Recent investigation with comparative serological assays (Boscia (1995)) demonstrated that the IIb component of cv. Chasselas 8/22 is the same as the GLRaV-2 isolate from France (Zimmermann (1990)) which also include the isolates of grapevine corky bark associated closteroviruses from Italy (GCBaV-BA) (Boscia (1995)) and from the United States (GCBaV-NY) (Namba et al., xe2x80x9cPurification and Properties of Closterovirus-like Particles Associated with Grapevine Corky Bark Disease,xe2x80x9d Phytopathology, 81:964-970 (1991) (xe2x80x9cNamba (1991)xe2x80x9d)). The IIa component of cv. Chasselas 8/22 was given the provisional name of grapevine leafroll associated virus 6 (GLRaV-6). Furthermore, the antiserum to the CA-5 isolate of GLRaV-2 produced by Boscia et al. (Boscia et al., xe2x80x9cCharacterization of Grape Leafroll Associated Closterovirus (GLRaV) Serotype II and Comparison with GLRaV Serotype III,xe2x80x9d Phytopathology, 80:117 (1990)) was shown to contain antibodies to both GLRaV-2 and GLRaV-1, with a prevalence of the latter (Boscia (1995)).
Several shorter closteroviruses (particle length 800 nm long) have also been isolated from grapevines. One of these, called grapevine virus A (xe2x80x9cGVAxe2x80x9d) has also been found associated, though inconsistently, with the leafroll disease (Agran et al., xe2x80x9cOccurrence of Grapevine Virus A (GVA) and Other Closteroviruses in Tunisian Grapevines Affected by Leafroll Disease,xe2x80x9d Vitis, 29:43-48 (1990), Conti, et al., xe2x80x9cClosterovirus Associated with Leafroll and Stem Pitting in Grapevine,xe2x80x9d Phytopathol. Mediterr., 24:110-113 (1985), and Conti et al., xe2x80x9cA Closterovirus from a Stem-pitting-diseased Grapevine,xe2x80x9d Phytopathology, 70:394-399 (1980)). The etiology of GVA is not really known; however, it appears to be more consistently associated with rugose wood sensu lato (Rosciglione at al., xe2x80x9cMaladies de l""enroulement et du bois strixc3xa9 de la vigne: analyse microscopique et sxc3xa9rologique (Leafroll and Stem Pitting of Grapevine: Microscopical and Serological Analysis),xe2x80x9d Rev. Suisse Vitic Arboric. Hortic., 18:207-211 (1986) (xe2x80x9cRosciglione (1986)xe2x80x9d), and Zimmermann (1990)). Moreover, another short closterovirus (800 nm long) named grapevine virus B (xe2x80x9cGVBxe2x80x9d) has been isolated and characterized from corky bark-affected vines (Boscia et al., xe2x80x9cProperties of a Filamentous Virus Isolated from Grapevines Affected by Corky Bark, Arch. Virol., 130:109-120 (1993) and Namba (1991)).
As suggested by Martelli I, leafroll symptoms may be induced by more than one virus or they may be simply a general plant physiological response to invasion by an array of phloem-inhabiting viruses. Evidence accumulated in the last 15 years strongly favors the idea that grapevine leafroll is induced by one (or a complex) of long closteroviruses (particle length 1,400 to 2,200 nm).
Grapevine leafroll is transmitted primarily by contaminated scions and rootstocks. However, under field conditions, several species of mealybugs have been shown to be the vector of leafroll (Engelbrecht et al., xe2x80x9cTransmission of Grapevine Leafroll Disease and Associated Closteroviruses by the Vine Mealybug Planococcus-ficus,xe2x80x9d Phytophylactica, 22:341-346 (1990), Rosciglione, et al., xe2x80x9cTransmission of Grapevine Leafroll Disease and an Associated Closterovirus to Healthy Grapevine by the Mealybug Planococcus ficus,xe2x80x9d (Abstract), Phytoparasitica, 17:63-63 (1989), and Tanne, xe2x80x9cEvidence for the Transmission by Mealybugs to Healthy Grapevines of a Closter-like Particle Associated with Grapevine Leafroll Disease,xe2x80x9d Phytoparasitica, 16:288 (1988)). Natural spread of leafroll by insect vectors is rapid in various parts of the world. In New Zealand, observations of three vineyards showed that the number of infected vines nearly doubled in a single year (Jordan et al., xe2x80x9cSpread of Grapevine Leafroll and its Associated Virus in New Zealand Vineyards,xe2x80x9d 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Montreux, Switzerland, pp. 113-114 (1993)). One vineyard became 90% infected 5 years after GLRaV-3 was first observed. Prevalence of leafroll worldwide may increase as chemical control of mealybugs becomes more difficult due to the unavailability of effective insecticides.
In view of the serious risk grapevine leafroll virus poses to vineyards and the absence of an effective treatment of it, the need to prevent this affliction continues to exist. The present invention is directed to overcoming this deficiency in the art.
The present invention relates to an isolated protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus. The encoding RNA and DNA molecules, in either isolated form or incorporated in an expression system, a host cell, or a transgenic Vitis or citrus scion or rootstock cultivar, are also disclosed.
Another aspect of the present invention relates to a method of imparting grapevine leafroll virus resistance to Vitis scion or rootstock cultivars by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus. These DNA molecules can also be used in transformation of citrus scion or rootstock cultivar to impart tristeza virus resistance to such cultivars.
The present invention also relates to an antibody or binding portion thereof or probe which recognizes the protein or polypeptide.
Grapevine leafroll virus resistant transgenic variants of the current commercial grape cultivars and rootstocks allows for more complete control of the virus while retaining the varietal characteristics of specific cultivars. Furthermore, these variants permit control of GLRaV transmitted either by contaminated scions or rootstocks or by GLRaV-carrying mealy bugs. With respect to the latter mode of transmission, the present invention circumvents increased restriction of pesticide use which has made chemical control of mealy bug infestations increasingly difficult. In this manner, as well as others, the interests of the environment and the economics of grape cultivation and wine making are all benefited by the present invention.